Establishment Of Agrobacterium-mediated Transient Expreesion System In Nicotiana Benthamiana, Salvia Miltiorrhiza And Prunella Vulgaris

Genetic transformation has always been an important method of studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it is time-consuming to obtain a stable transformed hairy root or transgenic plants, which is difficult to satisfy the great demand of researches on medical plant secondary metabolism related genes. Moreover, Agrobacterium-mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost and short period. However, presently, researches on medical plant functional genes commonly use stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation etc. Thus, in this study, we attempted to establish an Agrobacterium-mediated transient transformation system in Nicotiana benthamiana, Salvia miltiorrhiza Bunge and Prunella vulgaris L, aiming at providing a new method and reference for further study of their functional genes.In this study, we tried to build virus-induced gene silencing(VIGS) system in S. miltiorrhiza, to highly express the secondary metabolism biosynthesis-related genes and design a heterologous pathway of secondary metabolite synthesis with the pEAQ vector, and to establish a Agrobacterium-mediated transient transformation system in N. benthamiana, S. miltiorrhiza and P. Vulgaris seedling. The main conclusions were as follows:(1) In this experiment, we failed to establish the VIGS system in S. miltiorrhiza. This may be caused by the differences of Agrobacterium strains, bacterial infection concentration, growth status of the plant materials, virus strains and plant defense mechanisms against viruses etc. In addition, the cucumber mosaic virus(CMV) successfully identified from diseased leaves of S. Miltiorrhiza would offer materials and basis for building efficient viral silencing vectors.(2) One or more foreign genes in tobacco leaves were highly expressed through the pEAQ vector. This supplied a powerful method for the further study of its gene functions. Also, efficient co-expression of multiple foreign genes in tobacco made it possible to construct heterologous secondary metabolic pathways.(3) Agrobacterium-mediated transient transformation system showed different infection efficiencies in different plant seedling materials. In the test, none of the transient expression systems in S. Miltiorrhiza included VIGS, pEAQ mediated transient transformation and seedling transient transformation succeeded. This may be associated with the defense mechanisms against foreign genes in S. miltiorrhiza. The results of P. vulgaris and tobacco seedling transient transformation also suggested that the differences of the seedling instantaneous infecting efficiency may be related to differences in body size of plants, tissue density and other reasons.(4) Seedling instantaneous genetic transformation system was basically set up in P. vulgaris L by FAST and mechanical injury. This system provides an effective way to study functional genes in P. vulgaris. However, the system was not suitable for studying stress-related functional genes.